Protein-encapsulated oil particles

The This invention relates to an encapsulating oil particles with protein by complex coacervation and in particular an enzymatic Networking Proteineinkapselungshülle.

coacervation is the method by which an aqueous solution of a macromolecular Colloid into two liquid Phase is separated. A liquid Phase, which is referred to as the coacervate, consists of numerous very small colloid-rich droplets, which are bonded to each other. The other liquid phase, referred to as the equilibrium liquid is is an aqueous solution the coacervierenden agent.

If two or more oppositely charged macromolecular colloids be used to form the coacervate, the process is called complex coacervation. Colloids which a positive carry charge, include gelatin and agar. Colloids which a negative carry cargo, carboxymethyl cellulose and gum arabic include. Dependent on of the isoelectric point of each colloid, a dilution be necessary with water and / or an adjustment of the pH, that the special colloids are oppositely charged. These responses must at a temperature above the gelation temperature for the respective Colloid done, otherwise, the colloids are not in a liquid phase and a coacervation runs not from. If the coacervation takes place in an environment that oil particles contains, act the oil particles as a nucleating agent and the protein colloids deposit as hüllartige Structure around each oil particle from.

The Encapsulating oil particles in the process of complex coacervation is on the art well known. The<patcit><text>US 2800457 A</text></patcit> discloses oleaginous microscopic capsules and a process for producing same by complex coacervation. The<patcit><text>US 2800457 A</text></patcit> teaches a dispersing Colloid in water, the insertion of an oil, Forming an emulsion, dispersing a second colloid in Water, and mixing with the emulsion and adjusting the pH-value and / or diluting with water to form a complex coacervate, wherein all stages be carried out at a temperature above a gel point of the colloids, is carried out followed by cooling, to cause the formation of a gel, followed by the optional Stages of hardening connect and cross-linking with formaldehyde or an equivalent. in be an embodiment Gum arabic and gelatin to form a film of colloidal material hüllartigen an oil core around use. Once the coacervate was formed, the mixture is 1 hour at not more than 25 ° C ditched. After this time, the formation of the capsules is complete. The Capsules can then how required be used or they can the optional curing undergo. The<patcit><text>US 2800458 A</text></patcit> discloses a method in a similar manner for the manufacture of oleaginous Microcapsules. The<patcit><text>US 2800458 A</text></patcit> discloses the use of a saline solution for Formation of the coacervate while the <patcit><text>US 2800457 A</text></patcit> either a change the pH or diluting disclosed with water to form the coacervate.

A Crosslinking the protein shell the complex coacervate makes the thermostable protein encapsulated oil, as a protein having crosslinks is a stable structure. The use of known chemical crosslinking agents such as Formaldehyde or glutaraldehyde, to irreversibly linking the oleaginous Capsules on the relevant Art disclosed. Other crosslinking agents such as tannic acid (tannin) or potassium aluminum sulfate (alum) are similarly known. The both in the <patcit><text>US 2800 457 A</text></patcit> and in the <patcit><text>US 2800458 A</text></patcit> disclosed optional curing consists of a set of a suspension of capsular material to a pH value of 9 to 11, cooling to 0 ° C to 5 ° C and adding formaldehyde.

formaldehyde and glutaraldehyde are, even if they are effective chemical crosslinking agents, toxic. Thus oil capsules, which have been crosslinked using such chemicals, not be used for oils, on a body of a mammal are administered or taken up by this. This limits the applications for Such products, a strong.

Certain Naturally occurring enzymes are also good crosslinking agents. Such enzymes act to the extent that they determined the formation of bonds between Amino acid side chains catalyze in proteins. Since the enzymes are naturally occurring, sick beyond encapsulated oils, are enzymatically linked, not to a network with Formaldehyde and glutaraldehyde inherent problems and can therefore taken without concern of toxicity of the crosslinking agent or applied. As a cross-linking an enzyme-catalyzed Reaction is, must However, present the appropriate environmental conditions for optimal enzyme activity.

On Enzyme that catalyzes a protein cross-linking, is transglutaminase (Amine γ-glutamyltransferase, EC 2.3.2.13). Transglutaminase catalyzed acyl transfer reaction a between γ-carboxamide of glutamine residues in a peptide and various primary amines, often ε-amino groups of peptide-bound lysine residues. The&lt;?page 3?&gt;Result is a bond or cross-linking between a glutamine residue in a protein molecule and a Lysine residue in another protein molecule. For optimal activity requires Transglutaminase a divalent metal ion, usually calcium or magnesium, as a cofactor, and a pH value of about the 7th

The Japanese Patent Publication JP 5-292899 A Ajinomoto Inc. discloses the use of transglutaminase as crosslinking agents in the production of microcapsules. It will However, not assume that the in this patent publication structure taught a complex coacervate as defined by the skilled person on the relevant Art is. It's more of an enzyme-modified gelatin emulsion. About that also teaches the Japanese Patent Publication JP 5-292899 A crosslinking at elevated Temperatures. Molecule- and / or particulate structures that retain at elevated temperatures be, are more liquid and less stable, or to cross-linking of a molecule resulting from particles of undefined structure. The Ajinomoto publication "Ajinomoto Co.'s Tansglutaminase (TG) "discloses optimal networking conditions for transglutaminase at a pH 6-7 and increased Temperatures of 50 ° C.

article of the present invention is a process for the enzymatic encapsulation oil particles with protein by complex coacervation. According to this method, first, a complex coacervate above the gelation formed and an oil added and formed a coarse emulsion of oil particles in water. There is a cooling for Depositing a colloid shell the oil particles around and an enzyme is added to the water, the colloid shell at a temperature of 5 ° C to 10 ° C the formation of encapsulated oil particles by protein crosslinking enzyme.

The The method also achieves a number of advantages over the Techniques of the prior art. The process produces microcapsules with defined structures and sizes of various properties for different have end uses. For example, flavoring oils located in Proteineingekapselten particles ranging in size from 100 to 300 microns, sorted by size, to both to provide a significant flavor explosion when chewing, as well as to allow for processing in food applications. Although particle sizes greater than 300 microns can be formed, are such larger particles not suitable for spraying, extruding and other mechanical shear forces are required in many food applications. Furthermore are protein-encapsulated Geschmackstoffölteilchen thermostable and can a baking, frying and a treatment in the microwave resist.

in a preferred method according to the present Invention, first a coarse emulsion between the oil and the Colloidal dispersion of two oppositely charged colloids formed. Subsequently a complex coacervate with a protein shell around discrete oil particles formed around. The discrete particles are cooled to the surrounding protein shell to gel. The surrounding the discrete particles protein shell is subsequently at low temperatures in the formation of microcapsules of oil enzymatis CH crosslinked. It has been found that at low temperatures 20 ° C to 27 ° C, especially 5 ° C to 10 ° C, an enzymatic cross-linking in protein shells of fish and bovine gelatins achieved for providing the microcapsules of flavor oils can be. Further, the crosslinking reaction of these types of low temperatures do not pH-dependent. Thus, a wider pH range of from 2 to 10 or more are used, the number of the and the types of enzymes that can be used is widened.

in a preferred form of the present invention, transglutaminase for enzymatic cross-linking of the protein shell at a pH value of about 7 on a temperature range of 5 ° C to 10 ° C across used. Processing times and amounts of microencapsulated oils may for commercial Purposes in accordance with the preferred operating economically be achieved.

The Objectives and other advantages of the present invention can be with reference to the following figures, detailed description and the Example further understand.

<figref idrefs="S9">1</figref> is a photomicrograph a pre-emulsion of oil particles and colloids with 100x magnification.

<figref idrefs="S9">2</figref> is a photomicrograph one by aqueous dilution formed complex coacervate with 100x magnification.

<figref idrefs="S10">3</figref> is a photomicrograph encapsulated with protein oil particles, the slow cooling a complex coacervate formed at about 27 ° C, which is 100 Enlargement.

<figref idrefs="S10">4</figref> is a photomicrograph enzymatically encapsulated by protein oil particles in a final state at about 5 ° C with 100 × magnification.

how <figref idrefs="S9">1</figref> shows an oil (<figref>10</figref>) with a colloidal dispersion (<figref>12</figref>) Of at least one positive &lt;?page 4?&gt;loaded Protein colloid and at least one negatively charged colloid for Form a coarse emulsion stirred. In a preferred embodiment the positively charged protein colloid either gelatin or agar and the negatively charged colloid is either carboxymethyl cellulose, Sodium, gum arabic or a combination thereof. When gelatin is used, an amount of 10 wt .-% is preferred. A coarse emulsion of particles with a size ranging from 100 microns to 2,000 microns is formed.

how <figref idrefs="S9">2</figref> shows is a complex coacervate (<figref>14</figref>) At ambient temperature by aqueous dilution of the Colloid / oil emulsion educated. Dependent on from the isoelectric point of the protein colloid, adjusting the pH of the colloid / oil emulsion be used to form the complex coacervate.

The complex coacervate (<figref>14</figref>) Is heated to a temperature of the gel point the colloids or cooled below. how <figref idrefs="S10">3</figref> shows a Cool by sequentially cooling first to a temperature sufficient that the protein around each oil particle (<figref>10</figref>) Around in a football-shaped protein shell (<figref>16</figref>) separates, then by further cooling to stabilize the protein shell (<figref>16</figref>) Performed. As Alternative for cooling the protein may be denatured to the protein shell (<figref>16</figref>) to stabilize. Although the stabilization of the casing various ways may be achieved, cooling is preferably and it forms a distinct football-shaped protein shell (<figref>16</figref>) to the oil (<figref>10</figref>) Around. The extent of initial cooling depends on the gel point of the particular protein in the complex coacervate from. is example the gel point of fish gelatin from about 20 ° C while the gel point of bovine gelatin about 27 ° C is. would follow dependent on of the gelatin source an initial cooling a Cool to a temperature between 20 ° C and 27 ° C. The initial Cool is at a rate of about 1 ° C per 5 min performed. After the initial Cool, one football-shaped protein shell (<figref>16</figref>) to the complex coacervate (<figref>14</figref>) Around deposits, are The encapsulated oil particles with protein (<figref>18</figref>) Is further cooled to a temperature in a range from 5 ° C to 10 ° C. you be at 5 ° C to 10 ° C while a sufficient period of time held at the envelope protein (<figref>16</figref>) to stabilize.

how <figref idrefs="S10">4</figref> shows that chilled football-shaped protein shell is (<figref>16</figref>) enzymatically at 5 ° C linked to 10 ° C, a thermostable protein shell (<figref>20</figref>) to build. Transglutaminase is the preferred enzyme. you can of course occurring sources are obtained are chemically synthesized are produced or by using recombinant DNA methods. Transglutaminase is added to the complex coacervate in solution a carrier, such as dextrin, sodium or sugar added. The amount of is transglutaminase 1 wt .-% to 10 wt .-%. The support amount can be 99 wt .-% to 90 wt .-%. A divalent metal ion, preferably Calcium or magnesium, as a cofactor also available. Only very minimal amounts of calcium are needed, and such amounts are normally in the natural Tissue source for Transglutaminase present. Alternatively, the ion can be added, when it holds for an acceleration of the crosslinking reaction is required. Since transglutaminase an optimal activity has at a pH value of 7, the complex coacervate to cross-linking is the protein shell (<figref>20</figref>) Adjusted to a pH value of about the 7th

in a preferred embodiment, (please refer <figref idrefs="S9">1</figref>), A dispersion (<figref>12</figref>) Of gelatin and carboxymethylcellulose (in a weight ratio from 1: 0.1) with an oil (<figref>10</figref>) With stirring united. The obtained emulsified oil particles are washed with water diluted at ambient temperature, a complex coacervate (<figref>14</figref>) A gelatin shell around each oil particle to form around (see. <figref idrefs="S9">2</figref>). The gelatin is stabilized (gelled) to form a football-shaped shell (<figref>16</figref>) to the oil (<figref>10</figref>) Around (see. <figref idrefs="S10">3</figref>) by the temperature of the complex coacervate (<figref>14</figref>) With a rate of about 1 ° C per 5 min first to about 20 ° C to about 27 ° C and then rapidly to about 5 ° C to 10 ° C is reduced. Each encapsulated with protein oil particles (<figref>18</figref>) Has a size of about 100 to 300 microns. The gelled gelatin shell then with transglutaminase at a pH value of about 7 to form a thermostable capsules (<figref>20</figref>) Crosslinked (cf.. <figref idrefs="S10">4</figref>). The transglutaminase then by adjusting the capsule (<figref>20</figref>) With citric acid a pH of about less than 3 deactivated. The gelatin and the carboxymethylcellulose are in a ratio of 1: 0.1 before. These increased deactivation stage stability the capsules (<figref>20</figref>) And eliminates any gel formation at storage.

example 1

Deionized Water at 50 ° C preheated is, is for all rubber / gelatin solutions used. Carboxymethyl cellulose sodium salt (1.8631 g) and gum arabic RCC powder (0.1863 g) to water (91.1038 g) with vigorous stirring until full resolution added. The dispersion is cooled to 35 ° C to 40 ° C. Gelatin 250 Bloom Type A (18.6306 g) with deionized water (167.6758 g) stir until complete dissolve mixed. subsequently is the dispersion at 35 ° C to 40 ° C cooled. Without stirring, entered the rubber dispersion in the preemulsion tank and the foam is 15 to 20 minutes to disperse calmly. A defoamer may,&lt;?page 5?&gt;if necessary, be used.

A solution of 50% (w / w) sodium hydroxide or 50% (g / g), citric acid is added to deionized water (558.9196 g) in the Einkapselungsbehälter and the whole at 35 ° to 40 ° C heated. The stir is started again in the preemulsion. The desired flavor oil (149.0451 g) is slowly added to the combined gelatin / gum solution in the preemulsion was added and mixing carried out until the oil droplets, the desired own size. The pH is adjusted to a pH of 5.0 to 5.6. The pre-emulsion mixture is transferred to the dilution water in the Einkapselungsbehälter and subsequently to 25 ° C. at a rate of 1 ° C per 5 min slowly cooled. The batch is then rapidly from 25 ° C at 10 ° C chilled adjusted with sodium hydroxide to a pH of. 7

transglutaminase (10% active in dextrin) (0.23288 g) is slowly added to the batch. The Batch at 10 ° C for 16 h stirred. Stirring is subsequently stopped and the capsules are allowed to separate by flotation. About 48 to 50% of the water is drained from the bottom of the vessel, subsequently stirring is resumed and re-dispersed the concentrated capsules. A 10% (g / g) sodium benzoate solution (10.2469 g) is added to the Capsules added as a preservative. After thorough the batch is mixed with 50% citric acid to a pH value of 2.75 set and then 5 mixed to 10 min. A solution of xanthan gum (0.1% to 0.3%) and propylene glycol (0.2% to 0.6%) of slowly added to the mixture of the capsules to the viscosity of the capsules to stabilize and control. The mixing is continued for 30 min.